The present invention relates to a biological material enriched for components of mineralized cartilage tissue; a mineralized biological material; methods for producing the biological materials; and methods of using the biological materials.
Articular cartilage is a specialized tissue found at the end of articulating bons. It is responsible for the distribution of load resistance to compressive forces, and the smooth gliding that is part of joint function. Articular cartilage is joined to its underlying subchondral bone by a zone of calcified cartilage. The calcified cartilage zone is involved in the distribution of force across the joint. The calcified zone forms after secondary ossification and acts like a growth plate during maturation (Oegema Jr. T R and Thompson R C in Articular Cartilage and Osteoarthritis ed. Kuettner K. et al., pp. 319-331, 1992). Its thickness is in part a function of weight bearing as Muller-Gerbl et al. (J. Anat. 154:103, 1987) have shown. In that study they demonstrated that the percentage of cartilage consisting of calcified cartilage is constant and varies between individuals between 3 to 8.8% of the cartilage thickness (Quarto et al., J Cell Biol 110:1379, 1990).
Little is known about the composition of the calcified cartilage and the metabolism of chondrocytes within and immediately above the calcified cartilage zone. In vivo, the cells in this region have alkaline phosphatase activity. While the major collagen is type II, type X collagen is also present. Cartilage in the calcified zone contains about half the proteoglycans found within mature articular cartilage deep zones. Researchers have also reported the presence of osteonectin as well as a unique protein seen only in the calcified zone. The mineral present in the calcified zone varies between species, but shows the characteristic 1.67 calcium-to-phosphate ratio of hydroxyapatite (Oegema et al. in Articular Cartilage and Osteoarthritis, ed. Kuettner K. et al. pp. 319, 1992). The process of calcification has been found to be very complex, involving different interacting matrix molecules and careful regulation at the cellular level through a complex multi-component system which involves many cellular, hormonal, and physicochemical processes (Poole et al. Anat. Rec. 224:167, 1989).
Experimental studies have suggested that the calcified cartilage plays a role in the pathogenesis of the joint disease osteoarthritis. Remodelling of the calcified cartilage with reduplication of the tidemark (the interface between the calcified and non-calcified cartilage) is characteristic of osteoarthritis (Hough A J et al., in Arthritis and Allied Conditions, ed McCarty D J pp. 1571, 1989). It has been postulated that is possible that changes at the osteoarticular junction may contribute to the development of osteoarthritis. In support of this concept Thompson et al. (J. Bone Surg. 73A:990, 1991), using an acute transarticular damage dog model, observed that the dogs develop cartilage degeneration over time if the calcified cartilage and subchondral bone are damaged. This may be relevant to the pathogenesis of osteoarthritis in humans as MRI studies have shown that up to 72% of individuals, who injure their joints, will have subchondral fractures without cartilage damage (Vellet et al., Radiology 178:271, 1991).
Metabolic changes have been identified in osteoarthritis cartilage that suggest involvement of the deep and/or calcified cartilage in this disease. Increased numbers of matrix vesicles and matrix vesicle associated enzymes occur in osteoarthritic cartilage (Ali, S. Y. et al. Fed. Proc. 32:1494-1498, 1971; Lorenteon, R. et al., Acta. Orthop. Scand. 52:684, 1981; and, Einhorn T A et al, J. Orthop Research 3:160, 1985). In addition, chondrocytes isolated from osteoarthritic cartilage show increased synthesis of type X collagen (Walker et al. J. Orthop Research 13:4, 1995).
Study of the calcified cartilage zone has been hampered, in part, by the lack of an in vitro culture system. Although many types of in vitro mineralizing chondrocyte culture systems have been described, these cultures use either growth plate chondrocytes (Okihana H et al., Histochemical J 25:166, 1993; Nakagawa et al., Calcif. Tissue Int. 53:127, 1993; Kato Y., et al., Proc. Natl. Acad. Sci. USA 85: 9552, 1988), embryonic chondrocytes, or embryonic growth plate chondrocytes; cells whose function in vivo is to form bone (Glaser J H et al., Journal of Biological Chemistry, 256:12607, 1981; Gerstenfeld L C et al., Journal of Cell Biology, 112:501, 1993; Hascall V C et al., Journal of Biological Chemistry, 251:3511, 1976; Bruckner P et al., Journal of Cell Biology, 109:2537, 1989). The tissue formed by these cells is not well suited to the study of articular cartilage mineralization as the cartilage serves as a template for bone formation. The cells in the epiphyseal plate cartilage go through a series of cytological changes as they progress through to calcification. In addition, they are surrounded by small amounts of matrix which will be permeated by vascular channels (Poole et al. Anat. Rec. 224:167, 1989). In contrast, the calcified zone of adult cartilage appears to be hyaline cartilage that undergoes mineralization and does not usually undergo vascular invasion unless there is an underlying disease process. Therefore, it is not appropriate to extrapolate observations generated from these mineralizing cultures to the calcified articular cartilage.
U.S. Pat. No. 5,326,357 to the present inventor, describes a reconstituted cartilage tissue characterized by a continuous layer of cartilage tissue having a substantial extracellular matrix and possessing zones similar to those found in animal cartilage in vivo, and methods for preparing the reconstituted cartilage tissue.
The present inventor has generated an in vitro culture system which mimics the deep articular cartilage and adjacent calcified cartilage zone of articular cartilage. The cultured mineralized cartilaginous tissue contains calcium apatite mineral, matrix vesicles, Type X collagen and it has alkaline phosphatase activity. Polydisperse proteoglycans are synthesized by the chondrocytes in the mineralized cartilage tissue. The proteoglycans have a larger hydrodynamic size than the proteoglycans synthesized by articular chondrocytes in reconstituted non-mineralized cartilage in culture.
Broadly stated the invention relates to a biological material comprising a continuous layer of cartilaginous tissue reconstituted in vitro which contains components associated with cartilage mineralization. The invention also broadly contemplates a mineralized biological material characterized by having a biochemical composition and physiological organization substantially similar to the deep and contiguous calcified cartilage zones of articular cartilage found in animals in vivo.
Chondrocytes from the mid and superficial zones of articular cartilage tissue may be cultured on top of the biological material of the invention to produce a reconstituted mineralized cartilaginous tissue which comprises the mineralized biological material of the invention; and a mid and superficial non-mineralized layer adjacent to and contiguous with the mineralized biological material. The superficial and mid non-mineralized layers have a biochemical composition and physiological organization similar to the mid and superficial zones respectively, of articular cartilage found in animals in vivo. Therefore, the reconstituted mineralized cartilage tissue is substantially similar to articular cartilage tissue in vivo.
The invention also relates to a process for producing the biological material of the invention comprising isolating chondrocytes from the deep zone of cartilage tissue; forming a layer of the chondrocytes on a substrate, and; culturing the chondrocytes in growth media under suitable conditions so that the chondrocytes accumulate matrix and form cartilaginous tissue which is enriched with components associated with cartilage mineralization.
The process may additionally comprise the steps of culturing the chondrocytes or cartilaginous tissue in the presence of a mineralizing agent, to form a continuous layer of a mineralized biological material characterized by having a biochemical composition and physiological organization substantially similar to the deep and contiguous calcified cartilage zones of articular cartilage found in animals in vivo.
In the alternative, the process of the invention may optionally comprise the steps of culturing chondrocytes isolated from the mid and superficial zones of articular cartilaginous tissue on top of the cartilaginous tissue in the presence of a mineralizing agent to produce a reconstituted mineralized cartilage tissue which has a deep mineralized layer, and mid and superficial non-mineralized layers.
The invention further relates to a mineralized biological material comprising a continuous layer of mineralized cartilaginous tissue having a biochemical composition and physiological organization substantially similar to the deep and contiguous calcified cartilage zones of articular cartilage found in animals in vivo, obtained by (a) isolating chondrocytes from the deep zone of articular cartilage tissue; forming a layer of the chondrocytes on a substrate; and (b)(i) culturing the chondrocytes in growth media under suitable conditions so that the chondrocytes accumulate matrix and form cartilaginous tissue, and culturing the cartilaginous tissue in the presence of a mineralizing agent, or (ii) culturing the chondrocytes in growth media in the presence of a mineralizing agent.
The chondrocytes in the biological materials or reconstituted mineralized cartilaginous tissue may be transformed with recombinant vectors containing an exogenous gene encoding a biologically active protein which corrects or compensates for a genetic deficiency. Therefore, the invention also contemplates a mineralized biological material or reconstituted mineralized cartilaginous tissue wherein chondrocytes in the mineralized biological material or reconstituted mineralized cartilaginous tissue are transformed with recombinant vectors containing an exogenous gene encoding a biologically active protein which corrects or compensates for a genetic deficiency.
The invention still further relates to a system for testing a substance that affects calcification of articular cartilage tissue comprising: culturing a biological material, mineralized biological material, or reconstituted mineralized cartilaginous tissue of the invention in the presence of a substance which is suspected of affecting calcification, and determining the biochemical composition and/or physiological organization of tissue generated in the culture, with the biochemical composition and/or physiological organization of the biological material, mineralized biological material, or reconstituted mineralized cartilaginous tissue cultured in the absence of the substance. The substance may be added to the culture, or the chondrocytes in the biological materials or reconstituted mineralized cartilaginous tissue may be genetically engineered to express the substance i.e. the chondrocytes may serve as an endogenous source of the substance.
The invention still further relates to a method of using the biological materials and reconstituted mineralized cartilage tissue of the invention to test pharmaceutical preparations for efficacy in the treatment of diseases of the joint and to a method of using the biological materials and reconstituted mineralized cartilaginous tissue of the present invention as an implant to replace or repair damaged or deficient cartilage. The invention also contemplates using the biological materials of the invention in gene therapy.
The invention also contemplates a method of replacing or repairing damaged or deficient cartilage in a joint of a patient comprising implanting a mineralized biological material or reconstituted mineralized cartilaginous tissue of the invention in the joint of the patient. Methods for enhancing healing of a bone fracture in a patient are contemplated which comprise inserting a mineralized biological material or reconstituted mineralized cartilaginous tissue of the invention into the site of a fracture.